Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide variety of mammals including sheep and goats (scrapie), cervid spp. (chronic wasting disease), and humans (Creutzfeldt-Jakob disease). Our studies are focused on the prion protein (PrP) due to the critical role of this protein in controlling many aspects of TSE pathogenesis such as susceptibility to disease and interspecies transmission. A central event in TSE disease involves the conversion of the normal host cellular prion protein (PrPC) to a partially protease-resistant, aggregated, disease-associated isoform (PrPSc). TSE-induced pathology is usually associated with PrP-res deposition, but the mechanism of neurodegeneration is not understood. The nature of the infectious agent, called a prion, remains uncertain but is thought to be composed primarily of misfolded PrP, perhaps in complex with another host accessory molecule(s). PrPC is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, and the majority of PrPSc produced in vivo contains this GPI anchor. Membrane association of both normal and disease-associated PrP isoforms may influence many features of prion disease and PrPC function. Our work is focused on elucidating mechanisms of uptake, replication, and spread of prions, in addition to determining the biochemical composition of mammalian prions. In 2014, we have continued the characterization of novel synthetic strains of TSE agents that originate from purified recombinant PrP and further investigated mechanisms of uptake, propagation, and intercellular spread of PrPSc via imaging studies in novel neuronal culture systems. Experiments associated with the former line of investigation indicate we have isolated a unique synthetic TSE prion completely dependent on GPI-anchorless PrPC for propagation. We have also explored the role of non-neuronal brain cells in TSE infections and found evidence that fibroblasts and astrocytes may play an important role at early time points of infection due to enhanced uptake of PrPSc compared to neurons. In collaboration with Motohiro Horiuchi's lab, we have also shown that trafficking of input PrPSc from the late endosomal/lysosomal pathway to the endocytic recycling pathway is important for establishment of prion infection in cells.